The purpose of this project will be to increase our understanding of the physiologic mechanism of glucocorticoid hormone action in the AtT-20 mouse pituitary tumor cell. This project will further define the mechanism of entry into the target cell, investigate a possible new mechanism of antiglucocorticoid action, examine the mechanism and biologic significance of glucocorticoid binding and nuclear translocation in intact cells and characterize glucocorticoid receptor binding to the isolated and cloned ACTH gene. The membrane mechanism of glucocorticoid uptake by the AtT-20 cell will be investigated by preparing sealed, properly-oriented membrane vesicles which will then be used to study the kinetics of glucocorticoid uptake. Experiments will be done to determine whether glucocorticoid movement across the membrane can be stimulated by co-transport of ions such as sodium or by counter-transport of glucocorticoids thus indicating a carrier-mediated uptake mechanism. The ability of several glucocorticoids to cause nuclear translocation and their ability to compete for uptake by intact cells will be compared with their ability to suppress ACTH mRNA levels in AtT-20 cells. To assure comparative data the cells will be first adapted to grow in completely defined serumless medium to avoid effects of transcortin on steroid uptake. The mechanism of steroid-receptor interaction with the genome will be studied by cloning the ACTH gene from AtT-20 DNA and studying receptor binding to the purified, amplified gene. The significance of the ability of progesterone and other antiglucocorticoids to allosterically lower the affinity of the glucocorticoid-receptor complex will be determined by comparing the allosteric effect of these steroids with their ability to antagonize glucocorticoid suppression of ACTH mRNA levels.